Applied Bioinformatics Course [中文]

Sequence analysis of spider toxin and antifungal peptide

Structural analysis of Huwentoxin-I (1QK6), a neurotoxic peptide isolated from the venom of the Chinese bird spider Selenocosmia huwena [Qu et al., 1997] shows a structural feature of three disulfide bridges and three beta-strands. Sequence alignment and structure superposition of 1HWT with seven other peptides including funnel web spider toxin, cono-toxin and sweet taste repressor confirms that the core of this small folding unit is an anti-parallel beta-sheet and three disulfide bridges. This motif is one of the smallest folding unit belongs to the cystine-knot super family [Pallaghy et al., 1994; Harrison et al., 1996]. The connectivity of three disulfide bridges remains the same despite of the variation of sequence length and similarity. Careful comparison of our anti-fungal peptide against these 8 peptides implies that AFP may belong to this folding unit. A three dimensional model of AFP was constructed based on the above assumption.


A small cysteine-rich protein with antimicrobial activity was isolated from pokeweed (Phytolacca americana) seeds and purified to homogeneity. The protein inhibits the growth of several filamentous fungi and gram-positive bacteria. The protein was highly basic, with a pI higher than 10. The entire amino acid sequence of the protein was determined to be homologous to antimicrobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. americana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and sequenced. The deduced amino acid sequence shows the presence of a signal peptide at the amino terminus, suggesting that the protein is synthesized as a preprotein and secreted outside the cells. The chromosomal gene shows the presence of an intron located within the region encoding the signal peptide. Southern hybridization showed that there was small gene family encoding Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, and was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted into the environment of the seeds during germination, and may create an inhibitory zone against soil-borne microorganisms. The disulfide bridges of Pa-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicates that the protein has a small cystine-knot folding, a positive patch, and a hydrophobic patch.

Several NMR structures of anti-fungal peptide were reported in recent years. Their coordinates were deposited to the Brookhaven Protein Data Bank (PDB). The anti-fungal peptide (PDB code 1AFP) from Aspergillus giganteus consists of 51 amino acid residues among them are 12 Lysines. The activity of this peptide is the inhibition of the growth of a variety of filamentous fungi. It has no effect on the growth of mammalian cells, yeast and eubacteria. The structural feature of this peptide is a compact beta-barrel formed by five anti-parallel beta-strands and stabilized by four internal disulfide bridges [Campos-Olivas et al., 1995]. Interestingly, another anti-fungal peptide isolated from radish (Raphanus sativus L.) seeds (1AYJ) poses the same sequence length and same number of disulfide bridges as 1AFP. However, no sequence similarity exists between these two peptides and the folding unit of this peptide is not a beta-barrel, but a cysteine stabilized alpha-beta motif [Fant et al., 1998]. This motif of three anti-parallel beta-strands and an alpha-helix connected by three disulfide bridges has been found in the scorpion toxin family. The biological activity of 1AYJ reduces elongation of fungal hyphae and increases hyphal branching. A third peptide Drosomycin induced by the fruit fly Drosophila melanogaster exhibits a potent activity against filamentous fungi, but inactive against bacteria. This peptide is 7 residue shorter than the above two. Yet, the number of disulfide bridges remains the same. The NMR structure of this peptide (1MYN) reveals that it also belongs to the cysteine stabilized alpha-beta motif [Landon et al., 1997]. The anti-fungal peptide we reported seems not belong to the above two folding categories due to the small size in sequence length and the number of the disulfide bridges.

Materials and Methods

Table 1 shows the structure templates used in the modeling work. Seven templates were taken from the Brookhaven Protein Data Bank (PDB). Their PDB codes are 1AXH, 1AGG, 1EIT, 1VTX, 1OMN, 1OMG and 1GUR respectively. The structure of the Chinese bird spider toxin Huwentoxin-I (1HWT) was solved in our laboratory [Qu et al., 1997], which has not been deposited to the PDB bank. Sequence alignment was performed taking into account that three disulfide bridges are conserved among all these peptides (Fig. 1).

Table 1 Structure templates used for modeling







Funnel-web spider toxin

Insecticidal toxin



Chinese bird spider toxin

Neuromuscular transmission blocker



Funnel-web spider toxin

P-type calcium channel antagonist



Funnel-web spider toxin

Diverse ion channel specificity



Funnel-web spider toxin

Sodium channel blocker



Magus cone

P-type calcium channel antagonist



Magus cone

P-type calcium channel antagonist



Gymnema sylvestre

Sweet taste repressor

The model building was mainly carried on using the molecular modeling program Whatif [Vriend, 1998]. The NMR coordinates of 1AXH were used to build up the backbone fragments. Loops were searched against the Whatif built-in loop fragment database. The modeled structure was refined geometrically within Whatif and energy minimized with the CHARM program to reduce side chain crash.

Results and Discussion

Fig. 2 shows the model of the three dimensional structure of the anti-fungal peptide. The key feature of this model is the anti-parallel beta-sheet and the three disulfide bridges, which can be found in all the 8 templates. The two short strands Fig. 2 can be considered as a variation among different molecules.

The side chains of three basic residues Lys5, Lys36 and Arg38 located at one side of the molecule form a positive patch (top in Fig. 2) of the molecule. This implies the possible active site of this anti-fungal peptide as it was investigated by the mutational analysis that the basic amino acid residues contribute to the anti-fungal potency [Fant et al., 1998].

The side chains of three hydrophobic residues Phe25, Ile27 and Val34 sit at one side of the molecular surface (left side in Fig. 2), which is unusual in molecular packing. Interestingly, this anomalous hydrophobic surface was also found in the modeling study of the black-eyed pea trypsin inhibitor which belongs to the cysteine rich Bowman-Birk protease inhibitor family. The hydrophobic patch along one side of this inhibitor was explained as a packing force of the possible multimer arrangement of the protein by both theoretical and experimental study [de Freita et al., 1997]. Biological experiments are to be carried out on our anti-fungal peptide to explore understand this structural feature.

Superimposition of the constructed model onto 8 template shows the structural similarity of this anti-fungal peptide to all other templates (Fig. 3). The folding unit of these peptides belongs to the cysteine-knot super family. However, they are different from the anti-fungal peptide from radish seeds and (1AYJ) and Drosomycin (1MYN) which is featured by the cysteine stabilized alpha -beta motif. This modeling work, together with the NMR results from 1AFP, 1AYJ and 1MYN, suggests that different folding units of anti-fungal peptides may exist, though its evolutional basis is not fully understood.


Fig. 1 Sequence alignment of AFP on 8 templates

Six cystein residues which form the three conserved disulfide bridges in all these peptides are in bold face. The paring pattern of the disulfide bridges is indicated by lines at the top. Dashes denote amino acid residue deletion. The left most code in bold face at each line is the PDB code of the structure templates for modeling, except for 1HWT (see table 1). 2AFP is the anti-fungal peptide to be modeled. Number of amino acid residues of each peptide is shown at the right side of each line.

Fig. 2 The three-dimensional model of the anti-fungal peptide

The atoms in the constructed three-dimensional model are presented as sticks with the following color code: green for carbon, blue for nitrogen, red for oxygen, white for hydrogen and yellow for sulfide. A gray coil shows the backbone and the beta-strands are emphasized by red ribbons. The three disulfide bridges are in thick sticks with the sulfide atoms shown as balls.

Fig. 3 Structural superimposition of AFP on 8 templates

Structural superimposition of the anti-fungal model (2AFP) on 8 templates. C-alpha traces of each peptide are drawn in sticks with different colors. The side chains of disulfide bridges are shown as ball-stick in yellow. The PDB code (see table 1) of each chain is indicated with an arrow line.


3 spiders Pokeberry Pokeberry root 6 ICK mitif peptides 6 ICK mitif peptides
The pictures of spiders are from Liao-Zhi's thesis. The pictures of pokeberry are from the Internet.
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6 July 2024, J Luo, CBI, PKU, Beijing, China